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ABSTRACT: The Odd-skipped related genes Osr1 and Osr2 encode closely related zinc finger containing transcription factors that are expressed in developing limb. However, their role in osteoblast proliferation and differentiation remains controversial and little is known about their regulation. In this study we showed that both Osr1 and Osr2 were expressed in several murine and human osteoblast cell lines as well as in primary osteoblast cultures. Moreover, their transcript levels were regulated by a number of osteogenic stimuli in murine pre-osteoblast MC3T3-E1 cells. The most robust regulation of Osr1 and Osr2 mRNA levels was observed after stimulation with 1,25-dihydroxyvitamin D(3). 1,25-dihydroxyvitamin D(3) induced transcript levels of Osr1 and Osr2 and this up-regulation was confirmed in other osteoblast cultures, both from murine and human origin.
The Journal of steroid biochemistry and molecular biology 12/2012; · 2.66 Impact Factor
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ABSTRACT: Forkhead Box O (FoxO) transcription factors and Sestrins (SESN) are highly conserved and related stress-responsive proteins that protect the organism against age-related pathologies. For FoxOs, growing evidence shows a crucial role in osteoblast function. Here we investigated the role of different FoxO and SESN isoforms in 1,25(OH)(2)D(3)-treated MC3T3-E1 osteoblasts. 1,25(OH)(2)D(3) rapidly and strongly induced the expression of SESN1 and FoxO3a but down-regulated the expression of SESN3 and FoxO1. SESN2 and FoxO4 levels were hardly affected by 1,25(OH)(2)D(3). Chromatin Immunoprecipitation (ChIP)-sequencing revealed significant VDR/RXR binding to a DR3-type VDRE in SESN1 but not in the genomic region where FoxO3a is located. Mutation of the SESN1 VDRE abolished responsiveness to 1,25(OH)(2)D(3) in luciferase-based transfection assays. siRNA-mediated knock-down of SESN1, SESN3, FoxO1 or FoxO3a did not prevent 1,25(OH)(2)D(3) from reducing the expression of cell cycle markers like Cyclin D1 and Cdc6 and from exerting its characteristic antiproliferative effect on MC3T3-E1 osteoblasts. Accordingly, the 1,25(OH)(2)D(3)-induced reduction in the number of S-phase cells was also maintained. The antiproliferative effect was still present in primary osteoblast in which all three FoxO isoforms were deleted (TKOpOB). Interestingly, both MC3T3-E1 osteoblasts in which FoxO1 was knocked-down and TKOpOBs accumulated significantly more reactive oxygen species (ROS) after treatment with 1,25(OH)(2)D(3) than control cells. siRNA-mediated knock-down of individual SESN isoforms did not result in significant differences in ROS levels. In conclusion, 1,25(OH)(2)D(3) directly and indirectly alters the expression levels of different FoxO and SESN isoforms in osteoblasts, presumably not to exert its antiproliferative action but to control ROS levels. This article is part of a Special Issue entitled 'Vitamin D Workshop'.
The Journal of steroid biochemistry and molecular biology 09/2012; · 2.66 Impact Factor
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Carsten Kriebitzsch,
Lieve Verlinden, Guy Eelen,
Natasja M van Schoor,
Karin Swart,
Paul Lips,
Mark B Meyer,
J Wesley Pike,
Steven Boonen,
Carsten Carlberg,
Victor Vitvitsky,
Roger Bouillon,
Ruma Banerjee,
Annemieke Verstuyf
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ABSTRACT: High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY owing to its negative impact on collagen maturation. HCY is cleared by cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine preosteoblasts treated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)(2) D(3) . Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)(2) D(3) treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine, and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)(2) D(3) -treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knockout osteoblasts, in which upregulation of cbs in response to 1,25(OH)(2) D(3) was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D(3) levels] and HCY levels. In conclusion, this study showed that cbs is a primary 1,25(OH)(2) D(3) target gene which renders HCY metabolism responsive to 1,25(OH)(2) D(3).
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 08/2011; 26(12):2991-3000. · 6.04 Impact Factor
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Lieve Verlinden,
Annemieke Verstuyf, Guy Eelen,
Roger Bouillon,
Paloma Ordóñez-Morán,
María Jesús Larriba,
Alberto Muñoz,
Natacha Rochel,
Yoshiteru Sato,
Dino Moras,
Miguel Maestro,
Samuel Seoane,
Fernando Dominguez,
Silvina Eduardo-Canosa,
Daniel Nicoletti,
Edelmiro Moman,
Antonio Mouriño
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ABSTRACT: An improved synthetic route to 1α,25-dihydroxyvitamin D(3) des-side chain analogues 2 a and 2 b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF-7 breast cancer cells and prodifferentiation activity toward SW480-ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin D receptor (hVDR) complexed to one of these analogues, 20(17→18)-abeo-1α,25-dihydroxy-22-homo-21-norvitamin D(3) (2 a) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25-dihydroxyvitamin D(3).
ChemMedChem 05/2011; 6(5):788-93. · 3.15 Impact Factor
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ABSTRACT: Non-steroidal analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] represent a most particular class of analogs because they are either not directly derived from the core 1,25(OH)2D3-structure or they have modifications in the core structure that are so drastic that the steroidal structure is lost. Non-steroidal CD-ring analogs of 1,25(OH)2D3 have been developed to study the role of the central rigid CD-ring system in the biological activity of 1,25(OH)2D3. Here we review the different classes of CD-ring analogs and highlight some representative analogs such as the fluorinated D-ring analogs CD578, WU515 and WY1113 which show markedly increased differentiating activity on human SW480-ADH colon cancer cells, characterized by a stronger induction of the invasion suppressor E-cadherin and a stronger repression of the beta-catenin/TCF target oncogene c-Myc. Correspondingly, CD578, WU515 and WY1113 are more potent inhibitors of beta-catenin/TCF signaling than 1,25(OH)2D3 and induce stronger VDR-coactivator interactions. Underlying the increased biological potency of analog CD578 are additional contacts between the side chain fluorine atoms of the analog with specific residues of helix 12 (H12) of the Vitamin D Receptor (VDR) and subsequent stronger VDR-coactivator interactions.
The Journal of steroid biochemistry and molecular biology 07/2010; 121(1-2):417-9. · 2.66 Impact Factor
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Els Vanoirbeek, Guy Eelen,
Lieve Verlinden,
Kathleen Marchal,
Kristof Engelen,
Bart De Moor,
Ine Beullens,
Suzanne Marcelis,
Pierre De Clercq,
Roger Bouillon,
Annemieke Verstuyf
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ABSTRACT: 1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the biological active form of vitamin D. Its antiproliferative capacities make it a potential drug to treat diseases such as cancer. The clinical use of 1,25-(OH)2D3 as an antiproliferative agent is hampered by its calcemic effects. Hence, structural analogs such as the seco-9,11-bisnor-17-methyl analog, WY1112, have been developed with superagonistic capacities. This study aims to distinct the molecular activities of 1,25-(OH)2D3 and WY1112 and identify possible differences in gene expression.
Total RNA was extracted from MCF-7 breast cancer cells treated with 1,25-(OH)2D3 or WY1112 and was used for microarray analysis.
The experiments revealed that WY1112 induces the same genes as 1,25-(OH)2D3, but the induction level of the individual genes is higher. Microarray analysis did not reveal genes that were exclusively regulated by WY1112.
The superagonistic vitamin D analog WY1112 induces the same set of genes as 1,25-(OH)2D3, but the level of induction of the individual genes is higher.
Anticancer research 10/2009; 29(9):3585-90. · 1.73 Impact Factor
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ABSTRACT: The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an important regulator of bone metabolism, calcium and phosphate homeostasis but also has potent antiproliferative and pro-differentiating effects on a wide variety of cell types. To identify key genes that are (directly) regulated by 1,25(OH)2D3, a large number of microarray studies have been performed on different types of cancer cells (prostate, breast, ovarian, colorectal, squamous cell carcinoma and leukemia). The variety of target genes identified through these studies reflects the pleiotropic action of 1,25(OH)2D3. Common cellular processes targeted by 1,25(OH)2D3 in the different cancer cell lines include cell cycle progression, apoptosis, cellular adhesion, oxidative stress, immune function and steroid metabolism. Upon comparison of the lists of genes regulated by 1,25(OH)2D3 in the different microarray studies, only a small set of individual genes were commonly regulated, among which are included 24-hydroxylase, growth arrest and DNA-damage-inducible protein, cathelicidin antimicrobial peptide and multiple cyclins.
Anticancer research 10/2009; 29(9):3471-83. · 1.73 Impact Factor
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ABSTRACT: 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has potent antiproliferative actions but calcemic effects obstruct its application in the treatment of hyperproliferative disorders. Therefore, analogs of 1,25-(OH)2D3 are designed with a clear dissociation between both effects. Here the biological activity of the trans-decalin CD-ring analog CY10012 is discussed.
Proliferation/differentiation/transactivation assays as well as mouse models were used to determine the activity of CY10012 in vitro and in vivo.
CY10012, has ten-fold higher antiproliferative actions than 1,25(OH)2D3 but is also twice as calcemic. To determine the role of the Vitamin D Receptor (VDR) in mediating the calcemic actions of CY10012, the analog was daily administered to VDRwt and VDRko mice. This treatment caused drastic weight loss and death in VDRwt mice but not in VDRko mice.
Analog CY10012 has greater antiproliferative action but also two-fold higher calcemic effects which depended entirely on VDR-mediated signalling pathways.
Anticancer research 10/2009; 29(9):3579-84. · 1.73 Impact Factor
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ABSTRACT: Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of beta-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization.
Chemistry & Biology 11/2008; 15(10):1029-34. · 5.83 Impact Factor
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ABSTRACT: Vitamin D deficiency increases risk for type 1 diabetes in genetically predisposed individuals, while high doses of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] prevent insulitis and diabetes in NOD mice.
Since 1,25(OH)(2)D(3) regulates gene transcription through the vitamin D receptor (VDR), we investigated the role of VDR in diabetes development by creating NOD mice without functional VDR.
VDR(-/-) NOD mice are rachitic and have lower numbers of putative regulator cells [TCR-alpha/beta(+)CD4(-)CD8(-) (natural killer T-cells) and CD4(+)CD25(+) T-cells [in central and peripheral immune organs compared with VDR(+/+) NOD littermates. Lipopolysaccharide-stimulated VDR(-/-) NOD macrophages expressed lower interleukin (IL)-1, IL-6, and CC chemokine ligand 2 mRNA, correlating with less nuclear translocation of p65 nuclear factor-kappaB compared with VDR(+/+) NOD macrophages. Thymic and lymph node dendritic cells from VDR(-/-) NOD mice displayed an even less mature CD11c(+)CD86(+) phenotype than VDR(+/+) NOD mice. Despite this immune phenotype linked to diabetes in NOD mice, VDR(-/-) NOD mice developed insulitis and diabetes at the same rate and incidence as VDR(+/+) NOD littermates.
Despite aggravating known immune abnormalities in NOD mice, disruption of VDR does not alter disease presentation in NOD mice in contrast to the more aggressive diabetes presentation in vitamin D-deficient NOD mice.
Diabetes 02/2008; 57(1):269-75. · 8.29 Impact Factor
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Lieve Verlinden,
Isabelle Vanden Bempt, Guy Eelen,
Maria Drijkoningen,
Ilse Verlinden,
Kathleen Marchal,
Christiane De Wolf-Peeters,
Marie-Rose Christiaens,
Luc Michiels,
Roger Bouillon,
Annemieke Verstuyf
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ABSTRACT: We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D(3), a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER-/PR-/HER-2- phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present.
Cancer Research 08/2007; 67(14):6574-81. · 7.86 Impact Factor
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ABSTRACT: A previous cDNA microarray analysis in murine MC3T3-E1 osteoblasts revealed a cluster of genes involved in cell cycle progression that was significantly down-regulated after a single treatment with 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] [L. Verlinden, G. Eelen, I. Beullens, M. Van Camp, P. Van Hummelen, K. Engelen, R. Van Hellemont, K. Marchal, B. De Moor, F. Foijer, H. Te Riele, M. Beullens, M. Bollen, C. Mathieu, R. Bouillon, A. Verstuyf, Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3, J. Biol. Chem. 280 (45) (2005) 37319-37330]. Among those genes were the DNA replication and DNA damage checkpoint proteins, Chk1 and Claspin, of which the human homologues were recently shown to be E2F-responsive. Quantitative real-time PCR experiments in 1,25(OH)(2)D(3)-treated MC3T3-E1 cells confirmed the down-regulation observed in the microarray experiment. Moreover, Chk1 and Claspin promoter activities were also reduced after incubation with 1,25(OH)(2)D(3), and this reduction was mediated through the E2F recognition motifs within their promoters because mutation of these motifs almost completely abolished the repressive effect of 1,25(OH)(2)D(3). The antiproliferative effect of 1,25(OH)(2)D(3) as well as its potential to down-regulate the expression of Chk1 and Claspin depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) lost its antiproliferative action and failed to repress these E2F-target genes in p107(-/-);p130(-/-)-cells, but not in pRb(-/-)-cells.
The Journal of Steroid Biochemistry and Molecular Biology 04/2007; 103(3-5):411-5. · 3.05 Impact Factor
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ABSTRACT: 1,25-dihydroxy Vitamin D [1,25-(OH)(2)D] exerts its effects via the vitamin D receptor (VDR) that belongs to the steroid/thyroid hormone receptor superfamily leading to gene regulation which results in various biological responses. Within the last two decades, the receptor has been shown to be present not only in classical target tissues such as bone, kidney and intestine but also in many other non-classical tissues. Besides the almost universal presence of VDRs, some cell types (e.g. keratinocytes, monocytes, bone, placenta) are capable of metabolizing 25-hydroxyvitamin D to 1,25(OH)(2)D by the enzyme 1alpha-hydroxylase (CYP27B1). The combined presence of 25(OH)D-1alpha-hydroxylase as well as the specific receptor in several tissues introduced the idea of a paracrine role for 1,25(OH)(2)D. Moreover, it has been demonstrated that 1,25(OH)(2)D can induce differentiation and inhibit proliferation of a wide variety of cell types. The molecular mechanisms behind this antiproliferative action is thoroughly explored but the whole picture is still difficult to understand. Important cell cycle regulators are involved such as cyclins, cyclin dependent kinases and their corresponding inhibitors as well as E2F transcription factors and accompanying pocket proteins. However the precise hierarchical structure of this wide diversity of actions of 1,25(OH)(2)D on genes influencing cell cycle progression is not firmly established nor do we understand which pathways are essential and which redundant. The antiproliferative action makes 1,25-(OH)(2)D and its analogs a possible therapeutic tool to treat hyperproliferative disorders, among which different types of cancer. This review focuses on the effects of 1,25(OH)(2)D and its analogs on cell proliferation, the results in in vivo experiments in Vitamin D deficient or resistant animals to cancer and the current epidemiological and intervention studies linking Vitamin D status or treatment and human cancer.
The Journal of Steroid Biochemistry and Molecular Biology 01/2007; 102(1-5):156-62. · 3.05 Impact Factor
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Lieve Verlinden, Guy Eelen,
Ine Beullens,
Mark Van Camp,
Paul Van Hummelen,
Kristof Engelen,
Ruth Van Hellemont,
Kathleen Marchal,
Bart De Moor,
Floris Foijer,
Hein Te Riele,
Monique Beullens,
Mathieu Bollen,
Chantal Mathieu,
Roger Bouillon,
Annemieke Verstuyf
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ABSTRACT: 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has potent antiproliferative effects characterized by a hampered G(1)/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)(2)D(3). Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)(2)D(3) and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)(2)D(3)-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)(2)D(3) was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)(2)D(3) depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) failed to repress these E2F target genes and lost its antiproliferative action in p107(-/-);p130(-/-) cells but not in pRb(-/-) cells.
Journal of Biological Chemistry 12/2005; 280(45):37319-30. · 4.77 Impact Factor
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ABSTRACT: The non-classical effects of 1,25(OH)(2)D(3) create possible therapeutic applications for immune modulation (e.g. auto-immune diseases and graft rejection), inhibition of cell proliferation (e.g. psoriasis, cancer) and induction of cell differentiation (e.g. cancer). The major drawback related to the use of 1,25(OH)(2)D(3) is its calcemic effect, which prevents the application of pharmacological concentrations. Several analogs are now available that show modest to good selectivity with regard to specific effects (e.g. anticancer or immune effects or bone anabolism versus hypercalcemia) when tested in appropriate in vivo models. The molecular basis for this selectivity is only partially understood and probably a variable mixture of mechanisms.
The Journal of Steroid Biochemistry and Molecular Biology 11/2005; 97(1-2):21-30. · 3.05 Impact Factor
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ABSTRACT: The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH)(2)D(3) in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH)(2)D(3) namely 19-nor-14-epi-23-yne-1,25-(OH)(2)D(3) (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)(2)D(3) (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH)(2)D(3). Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR-coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)(2)D(3). TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR-DRIP205 interaction induced by 1,25-(OH)(2)D(3) at 10(-8)M. Evaluation of additional 1,25-(OH)(2)D(3)-analogs and their impact on VDR-coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR-coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.
The Journal of Steroid Biochemistry and Molecular Biology 11/2005; 97(1-2):65-8. · 3.05 Impact Factor
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ABSTRACT: Two 14-epi-analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)(2)D(3)], 19-nor-14-epi-23-yne-1,25-(OH)2D3 (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)2D3 (TX527), show enhanced antiproliferative (at least 10-fold) and markedly lower calcemic effects both in vitro and in vivo, compared with 1,25-(OH)2D3. This study aimed to evaluate their superagonistic effect at the level of interaction between the Vitamin D receptor (VDR) and coactivators. Mammalian two-hybrid assays with VP16-fused VDR and GAL4-DNA-binding-domain-fused steroid receptor coactivator 1 (SRC-1), transcriptional intermediary factor 2 (Tif2), or DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)2D3 (up to 16- and 20-fold stronger induction of VDR-SRC-1 interaction for TX522 and TX527 at 10(-10) M). Similar assays in which metabolism of 1,25-(OH)2D3 was blocked with VID400, a selective inhibitor of the 1,25-(OH)2D3-metabolizing enzyme CYP24, showed that the enhanced potency of these analogs in establishing VDR-coactivator interactions can only partially be accounted for by their increased resistance to metabolic degradation. Crystallization of TX522 complexed to the ligand binding domain of the human VDR demonstrated that the epi-configuration of C14 caused the CD ring of the ligand to shift by 0.5 angstroms, thereby bringing the C12 atom into closer contact with Val300. Moreover, C22 of TX522 made an additional contact with the CD1 atom of Ile268 because of the rigidity of the triple bond-containing side chain. The position and conformation of the activation helix H12 of VDR was strictly maintained. In conclusion, this study provides deeper insight into the docking of TX522 in the LBP and shows that stronger VDR-coactivator interactions underlie the superagonistic activity of the two 14-epi-analogs.
Molecular Pharmacology 06/2005; 67(5):1566-73. · 4.88 Impact Factor
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ABSTRACT: The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH)(2)D(3) in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH)(2)D(3) treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12h after treatment with 1,25-(OH)(2)D(3) (10(-8)M), preceding (6h) or coinciding with (12h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12h incubation period with 1,25-(OH)(2)D(3) were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH)(2)D(3) was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH)(2)D(3) resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest.
The Journal of Steroid Biochemistry and Molecular Biology 06/2004; 89-90(1-5):405-7. · 3.05 Impact Factor
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ABSTRACT: To identify key genes in the antiproliferative action of 1,25(OH)2D3, MC3T3-E1 mouse osteoblasts were subjected to cDNA microarray analyses. Eleven E2F-driven DNA replication genes were downregulated by 1,25(OH)2D3. These results were confirmed by quantitative RT-PCR in different cell types, showing the general nature of this action of 1,25(OH)2D3.
1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] has a potent antiproliferative action characterized by a blocked transition from the G1- to the S-phase of the cell cycle. This study aims to identify genes whose expression is markedly altered after 1,25(OH)2D3 treatment in parallel with or preceding the observed G1-arrest.
The cDNA microarray technique was used, and the expression of approximately 4600 genes in MC3T3-E1 mouse osteoblasts was studied 6 and 12 h after treatment with 10(-8) M 1,25(OH)2D3. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed on MC3T3-E1 cells and on wildtype and vitamin D receptor (VDR) knockout primary murine epidermal keratinocytes (VDRwt MEKs, VDR-/- MEKs) and murine mammary tumor cells (GR) to confirm the microarray data.
After 12 h of treatment, in parallel with the 1,25(OH)2D3-induced G1 arrest, a particular set of DNA replication genes including a cell division cycle 6 homolog, a DNA polymerase alpha subunit, proliferating cell nuclear antigen, two DNA polymerase delta subunits, and flap-structure specific endonuclease 1, was downregulated at least 2-fold. These genes are known targets of the E2F family of transcription factors, which are probably the central mediators of this action of 1,25(OH)2D3. Indeed, as shown by transfection assays with an E2F reporter construct, 12- and 24-h treatment of MC3T3-E1 cells with 1,25(OH)2D3 reduced E2F activity by 49% and 73%, respectively. Quantitative RT-PCR analyses confirmed the downregulation of these DNA replication genes by 1,25(OH)2D3 in MC3T3-E1, GR, and VDRwt MEKs cells, but not in VDR-/- MEKs cells, showing that this 1,25(OH)2D3-driven antiproliferative action is of a general nature and depends on a functional VDR.
Journal of Bone and Mineral Research 02/2004; 19(1):133-46. · 6.37 Impact Factor
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Lieve Verlinden,
Annemieke Verstuyf,
Christel Verboven, Guy Eelen,
Camiel De Ranter,
Ling-Jie Gao,
Yong-Jun Chen,
Ibrahim Murad,
Mihwa Choi,
Keiko Yamamoto,
Sachiko Yamada,
Dirk Van Haver,
Maurits Vandewalle,
Pierre J De Clercq,
Roger Bouillon
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ABSTRACT: Deletion of C19 in the structure of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] does not substantially alter the biological potency but prevents the conversion between the vitamin and the previtamin form. Hence, this modification allows the study of locked previtamin and vitamin forms. The locked 19-nor-1,25(OH)2-previtamin D3 analog (19-nor-previtamin D) had a low biological activity and was a rather weak activator of the genomic signal transduction pathway. 19-Nor-trans-decalin-1,25(OH)2-vitamin D3 (19-nor-TD-vitamin D), characterized by the presence of a trans-fused decalin CD-ring system, was 10-fold more potent than the parent compound and was a potent activator of the genomic signal transduction pathway. Surprisingly, the previtamin, 19-nor-trans-decalin-1,25(OH)2-previtamin D3 (19-nor-TD-previtamin D), was as potent as 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation and represents the first previtamin structure with pronounced vitamin D-like activity. Furthermore, this compound interacted as efficiently as 1,25(OH)2D3 with the vitamin D receptor (VDR), retinoid X receptor (RXR), coactivators, and DNA, which illustrated its potent ability to activate the genomic signal transduction pathway. Analysis of the transactivation potency of 12 VDR point mutants after stimulation with 19-nor-TD-previtamin D revealed that this analog used the same contact points within the receptor as did 1,25(OH)2D3. This could be confirmed by modeling analysis of this compound in the ligand binding pocket of VDR. In conclusion, a previtamin D3 analog is presented with genomic activities equivalent to 1,25(OH)2D3.
Journal of Biological Chemistry 10/2003; 278(37):35476-82. · 4.77 Impact Factor
